CN101704886B

CN101704886B - Human alpha-defensin 5 antiviral active mutant polypeptide and preparation method and application

Info

Publication number: CN101704886B
Application number: CN2009101914856A
Authority: CN
Inventors: 王军平; 陈芳; 王艾平; 申明强; 王崧; 粟永萍; 程天民
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2009-11-17
Filing date: 2009-11-17
Publication date: 2012-03-21
Anticipated expiration: 2029-11-17
Also published as: WO2011060613A1; CN101704886A; WO2011060613A9

Abstract

The invention relates to human alpha-defensin 5 (HD5) antiviral active mutant polypeptide and preparation method and application. The 21th glutamic acid in HD5 of the amino acid sequence of the mutant polypeptide can be replaced into arginine (E21R). The preparation method of the mutant polypeptide has two preparation methods, namely a solid-phase chemical synthesis method and a gene engineering preparation method. The HD5 mutant polypeptide of the invention has efficient antiviral activity under the conditions that serum exits or absents, and compared with parent HD5 polypeptide, the antiviral function of the invention is obviously improved. The HD5 mutant polypeptide of the invention is used for preventing viral infection, such as HSV, HPV, HIV and the like.

Description

People's α-alexin 5 antiviral active mutant polypeptides and preparation method and application

Technical field

The invention belongs to protein engineering and antiviral preparing technical field, particularly a kind of people's α-alexin 5 antiviral active mutant polypeptides.

Background technology

Disease of viral infection serious harm human health, development is efficient, the specificity antivirus medicine has important practical significance.

Alexin a kind of molecular weight that to be people find in the process of defence such as research Mammals, insect and invasion and attack of opposing pathogenic micro-organism and infection is the endogenous cationic peptide of 3-6kD.Why being called as cationic peptide, be because sophisticated alexin intramolecularly is rich in l-arginine (a kind of basic aminoacids), thereby the alexin molecule all has stronger positive charge as a rule.People are based on its quick, strong fungicidal activity of imitating to alexinic initial understanding, but discover afterwards that except anti-microbial activity, most of alexin also had antimycotic, anti-spirochete and antiviral effect.Especially some alexins demonstrate certain killing and wounding and restraining effect to having a strong impact on the virus of human health like hsv (HSV), human papilloma shape virus (HPV), human immunodeficiency virus (HIV) etc.Research data shows that the alexin molecule can be brought into play antivirus action through links such as the obstruction virion adhere to and invades mammalian cell and suppresses that viral DNA is transcribed.Because alexin is a kind of endogenous molecule, itself have the ability of identification pathogenic micro-organism and organism normal cell, so in effective dosage ranges, can not cause obvious influence, can not bring out producing the resistance phenomenon to the human normal cell yet.Just because of this, alexinic development has broad application prospects aspect preventing and treating at cause pathogeny imcrobe infection with exploitation.

People's α-alexin 5 (HD5) is mainly by intestinal epithelial cell and reproductive tract mucomembranous epithelial cell secreting, expressing.Because it is viral at the intravital main propagation of people position that the reproductive tract mucous membrane is HIV, HPV and HSV-2 etc., therefore, HD5 should have more anti-this type of virus activity theoretically.Buck etc. find when carrying out the anti-HPV of α alexin research, compare with other known human alexin molecule, the antiviral activity of HD-5 the most outstanding (Buck CB et al, PNAS, 2006,103:1516-1521).But, any alexin no matter, it is remarkable that its antivirus action all is not so good as antibacterial effect; Research shows general (the Tanabe Hetal more than the high several times of IC50 when antibiotic of medium effective concentration (IC50) when the α alexin is antiviral; J Virol, 2004,78:11622-11631).This just means that its consumption will significantly increase when application alexin polypeptide carries out antiviral therapy, so not only can add heavy patient's economical load, the more important thing is to exceed the tolerance of human normal cell to it, causes toxic side effects.In addition, the most of alexin polypeptide that comprise HD5 are under the situation that serum exists, and its antiviral activity can receive remarkable inhibition, thereby make interior application of alexinic body be very limited.

Known, alexinic antibiotic and antiviral activity is not only relevant with himself peculiar bipolarity, but also with whole molecule and intramolecule critical positions amino-acid residue positively charged what relevant.There is research to confirm, when the l-arginine that the alexin intramolecularly is positively charged (basic aminoacids) when replacing with neutral amino acids or acidic amino acid, alexinic antibioticly can significantly descend with antiviral activity.Otherwise; When if a certain or some acidic amino acid sports basic aminoacids with the alexin intramolecularly, the antibiotic and antiviral activity of the alexin molecule after the sudden change might be significantly improved (Higgs Retal, Immunogenetics; 2007,59:573-580).

Summary of the invention

The purpose of this invention is to provide a kind of people's α-alexin 5 antiviral active mutant polypeptides and preparation method and application.According to change alexin intramolecularly amino-acid residue form with electrically charged amount can influence the characteristic of its biological function; Have at HD5 self on the basis of virus functions such as certain anti-HSV, HPV and HIV; Through critical positions amino acid rite-directed mutagenesis and antiviral activity screening, obtain the remarkable enhanced HD5 of antiviral activity mutant polypeptide.HD5 mutant polypeptide of the present invention all has antiviral activity efficiently under the situation that has serum-free to exist, the more maternal HD5 polypeptide of its antivirus action significantly improves.HD5 mutant polypeptide of the present invention can be used to prevent and treat the infection of viruses such as HSV, HPV and HIV.

Technical scheme of the present invention is:

The aminoacid sequence of people's α-alexin 5 antiviral active mutant polypeptides is following:

Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser

1 5 10 15

Leu Ser Gly Val Cys Arg Ile Ser Gly Arg Leu Tyr Arg Leu Cys

20 25 30

Cys Arg

The 21st L-glutamic acid of natural HD5 (Glu, E) be replaced by l-arginine (Arg, R).

The dna sequence dna of coding power people α-alexin 5 antiviral active mutant polypeptides, its sequence is following:

GCC ACC TGC TAT TGC CGA ACC GGC CGT TGT GCT ACC CGT GAG TCC CTC TCCGGG GTG TGT

ATC AGT GGC CGC CTC TAC AGA CTC TGC TGT CGC

The preparation method of HD5 mutant polypeptide comprises solid state chemistry synthesis method and genetically engineered preparation method.

Gene engineering research prepares the method for HD5 mutant polypeptide, and the dna sequence dna of above-mentioned people's α-alexin 5 antiviral active mutant polypeptides is cloned in the expression vector, and transfection host cell through abduction delivering, purifying, promptly gets said HD5 mutant polypeptide.

Described expression vector is a plasmid expression vector.

Described host cell comprises prokaryotic cell prokaryocyte and eukaryotic cell.

Said prokaryotic cell prokaryocyte is a Bacillus coli cells.

Said eukaryotic cell is a yeast cell.

People's α-alexin 5 antiviral active mutant polypeptides are in prevention and treat the application that hsv (HSV) infects or human papilloma shape virus (HPV) infects or human immunodeficiency virus (HIV) infects.This HD5 mutant polypeptide can be used separately, also can use with other drug or preparation compatibility.

The host cell of above-mentioned expression HD5 mutant polypeptide of the present invention can be bacterium, yeast and mammalian cell etc., wherein pichia spp preferably.

The method of purifying HD5 mutant polypeptide of the present invention comprises slightly to be carried, saltouts and LC etc., and wherein LC has comprised IX, chromatographic technique such as hydrophobic again.

HD5 mutant polypeptide of the present invention has the ability of viruses such as outstanding anti-HSV-2, HSV-1 and HPV.

HD5 mutant polypeptide of the present invention has significant antivirus action equally in 2%, 10% and 20% serum, the more maternal HD5 polypeptide of its antiviral activity significantly improves.

HD5 mutant polypeptide of the present invention does not have significant cytotoxicity.

The characteristics of HD5 mutant polypeptide of the present invention are; On the basis that keeps the basic biological characteristics of HD5 (for example: have no side effect, can not induce virus to produce resistance etc.) to the human normal cell; The HD5 mutant polypeptide has not only significantly improved antiviral ability; And the influence that not existed by serum of its antiviral activity, thereby make this mutant polypeptide not only can be used as externally applied agent but also can be used as the control that innerlich anwenden is applied to diseases of viral infection such as HSV, HPV and HIV.

Description of drawings:

Fig. 1 is that the HD5 mutant polypeptide suppresses the analysis that HSV-2 infects the vero cell effect under the serum-free situation, and wherein, column 1 is represented the normal cell contrast; Column 2 is represented virus control; Column 3 is represented the contrast of HD5 polypeptide, and column 4 is represented the HD5 mutant polypeptide, *: p＜0.01 vs. virus control;

Fig. 2 is that the HD5 mutant polypeptide suppresses the analysis that HSV-2 infects the vero cell effect under 2% serum condition, and wherein, column 1 is represented the normal cell contrast; Column 2 is represented virus control; Column 3 is represented the contrast of HD5 polypeptide, and column 4 is represented the HD5 mutant polypeptide, *: p＜0.01 vs. virus control; #:p＜0.01vs.HD5 polypeptide contrast;

Fig. 3 is that the HD5 mutant polypeptide suppresses the analysis that HSV-2 infects the vero cell effect under 10% serum condition, and wherein, column 1 is represented the normal cell contrast; Column 2 is represented virus control; Column 3 is represented the contrast of HD5 polypeptide, and column 4 is represented the HD5 mutant polypeptide, *: p＜0.01 vs. virus control; #:p＜0.01vs.HD5 polypeptide contrast;

Fig. 4 is that the HD5 mutant polypeptide suppresses the analysis that HSV-2 infects the vero cell effect under 20% serum condition, and wherein, column 1 is represented the normal cell contrast; Column 2 is represented virus control; Column 3 is represented the contrast of HD5 polypeptide, and column 4 is represented the HD5 mutant polypeptide, *: p＜0.01 vs. virus control; #:p＜0.01vs.HD5 polypeptide contrast.

Embodiment

Further specify the bright essentiality content of we with embodiment below, but content of the present invention is not limited thereto.

Main agents and material

1.Pyrobest ^TMDNA Polymerase test kit (TaKara Company products, DaLian, China)

2. restriction endonuclease EcoR I, SacI (New England BioLabs Company products, the U.S.)

3. plasmid vector pPIC9K, Pichi strain GS115 (Invitrogen Company products, the U.S.)

4.Red/ET recombinase red γ/red β/red α/redA plasmid pSC101-BAD-gbaA, intestinal bacteria HS996 (Gene Bridges Company products, Germany)

5.L-Arabic polysaccharide (Sigma Company products, the U.S.)

6.CCK-8 cell proliferation/toxicity detection kit (DOJINDO Company products, Japan)

7.Vero cell strain (preserve this chamber)

8.HSV-2 virus strain (preserve this chamber)

9.G418, penbritin (Roche Company products, Germany)

10.DMEM substratum, foetal calf serum (Hyclone Company products, the U.S.)

11.SP chromatography medias (GE Company products, the U.S.) such as Sepharose Fast Flow, Source 15RPC

12.C18 reversed phase chromatography, FINELINE PILOT 35 chromatography columns (GE Company products, the U.S.)

13. yeast extract, Tryptones, no amino acid yeast nitrogen (Oxford Company products, the U.S.)

14.LB substratum

Yeast extract 5g

Tryptones 10g

NaCl 10g

Be dissolved in the 1000ml deionized water, and regulate pH value to 7.0, autoclaving with the NaOH of 1mol/L.

15.YPD liquid nutrient medium

Yeast extract 2g

Tryptones 4g

Be dissolved in the 180ml deionized water autoclaving.It is the 100mg/ml penbritin that cooling back adds 20% Vadex and the 0.2ml concentration of 20ml after the filter degerming.

16.BMGY liquid nutrient medium

Yeast extract 10g

Tryptones 20g

No amino acid yeast nitrogen 13.4g

Glycerine 10g

Potassiumphosphate 26.631g

Be dissolved in 1000ml distilled water mesohigh sterilization, being chilled to and adding vitamin H to final concentration after the room temperature is 4 * 10 ^-5/ L, penbritin final concentration are 100 μ g/ml, regulate pH to 6.0, and 4 ℃ of preservations are subsequent use.

17. phosphate buffered saline buffer

NaCl 8g

KCl 0.2g

Na ₂HPO ₄ 1.44g

KH ₂PO ₄ 0.24g

Be dissolved in the 1000ml deionized water, and regulate pH value to 7.4, autoclaving with dense HCl.

The mechanochemical method of embodiment 1 HD5 mutant polypeptide is synthetic:

1, according to the aminoacid sequence of HD5 two mutants, (433A AppliedBiosystem) entrusts Shanghai to give birth to the synthetic HD5 mutant polypeptide of worker bio-engineering corporation to utilize full-automatic polypeptide synthetic instrument.

2, through HPLC anti-phase C18 column chromatography, desalination synthetic HD5 mutant polypeptide is carried out purifying.

3, adopt, purifying maternal HD5 polypeptide reference substance synthetic with quadrat method.

4, for the HD5 mutant polypeptide of purifying; Adopting high-pressure liquid phase (HPLC) method to carry out purity identifies; Using ground substance assistant laser desorption ionization time-of-flight mass spectrometry (TOFMS) (MALDI-TOF) measures its molecular weight; Measure its iso-electric point through isoelectric focusing electrophoresis, measure the aminoacid sequence structure with automatic Protein Sequencer.

5, HPLC result shows, purity＞95% of final gained HD5 mutant polypeptide; The isoelectric focusing electrophoresis result shows that the iso-electric point of gained HD5 mutant polypeptide is about 9.2; Mass spectrometry results shows that the molecular weight of gained HD5 mutant polypeptide is 3615.27, and is all consistent with the theoretical derivation value.

Expression and the purifying preparation of embodiment 2 reorganization HD5 mutant polypeptides in pichia spp

1, the full-length gene of composite coding HD5 two mutants.Entrust Shanghai to give birth to the full-length gene of the worker composite coding HD5 of bio-engineering corporation two mutants, gene order is following:

GCC ACC TGC TAT TGC CGA ACC GGC CGT TGT GCT ACC CGT GAG TCC CTC TCCGGG GTG TGT ATC AGT GGC CGC CTC TAC AGA CTC TGC TGT CGC

Figure DEST_PATH_GA20186499200910191485601D00012

Annotate: 3 coded amino acid of base of black matrix part are the l-arginine after the sudden change, and what 3 bases in the square frame were coded is terminator codon.

2, the structure of reorganization HD5 two mutants Yeast expression carrier

(1) PCR design of primers: in order to guarantee the no unnecessary amino acid of reorganization HD5 two mutants N end of yeast expression, will the encode gene order of HD5 two mutants of employing Red/ET recombinant technology directly is inserted into the STE among the Yeast expression carrier pPIC9K ₁₃After the proteolytic enzyme recognition site.For this reason, the at first synthetic upstream and downstream PCR primer that has the 50bp homology arm, primer sequence is following:

mHD5-up：5′

Figure DEST_PATH_GA20186499200910191485601D00013

Figure DEST_PATH_GA20186499200910191485601D00014

-3′

mHD5-down：5′

Figure DEST_PATH_GA20186499200910191485601D00015

Figure DEST_PATH_GA20186499200910191485601D00016

-3′

Wherein 5 ' end italicized item is STE in the pPIC9K plasmid among the mHD5-up ₁₃The sequence of 50 bases before the proteolytic enzyme recognition site, black matrix partly is the specificity upstream primer sequence of HD5 mutant gene; 5 ' end italicized item is the sequence of 50 bases after the MCS in the pPIC9K plasmid among the mHD5-down, and black matrix partly is the specificity downstream primer sequence of HD5 mutant gene.Carrying out the pcr amplification coding HD5 segmental while of mutant gene like this, its two ends have loaded respectively and the homeologous 50bp sequence of pPIC9K plasmid vector skeleton.

(2) pcr amplification: with synthetic HD5 two mutants full-length gene dna fragmentation is template, as the upstream and downstream primer, carries out the PCR reaction with mHD5-up and mHD5-down, and reaction conditions is following: 1. sex change: 94 ℃, and 5min; 2. sex change: 94 ℃, 30s; 3. renaturation: 55 ℃, 30s; 4. extend: 72 ℃, 30s; 5. return step " 2. ", 35 circulations; 6. extend: 72 ℃, 5min; 7. 4 ℃ of preservations, the global cycle number of times is 35 times.The PCR product is carried out 1% agarose gel electrophoresis, and the result shows the DNA band that amplifies about 150bp size.Gained PCR product is the HD5 mutant gene fragment that two ends have the 50bp homology arm respectively.

(3) structure of Red/ET mediation reorganization HD5 two mutants Yeast expression carrier: the pPIC9K fragment and the above-mentioned pcr amplification product of EcoR I enzyme tangent typeization are hit the HS996 competent cell that 0.1%L-pectinose abduction delivering red γ/red β/red α/redA recombinase has been used in conversion by the equal concentrations common-battery; Be inoculated in the LB flat board then; 37 ℃ of overnight cultures; Picking is cultivated through the positive colony of kantlex screening again; And extract plasmid and carry out the enzyme evaluation of cutting and check order, the result shows that the plasmid sequence of acquisition is correct, positive colony called after pPIC9K-HD5-E21/R.

The pPIC9K-HD5-E21/R DNA that order-checking is correct transforms the GS115 competent cell respectively after receiving with the switchback of Sac I enzyme.Electric shock transforms parameter: 1500V, 200 Ω, 25 μ F.Then transformed bacteria liquid is inoculated in the YPD flat board, cultivates identify the clone strain that is integrated with goal gene through genome PCR, prepare competent cell, shocking by electricity once more by preceding method transforms and identifies.Then carry out phenotype and G418 resistance screening, finally pick out clone strain inoculation culture, and identify frozen in-70 ℃ of guarantor's kinds with His+/Mut+ phenotype and tolerance 5.0mg/ml G418 with ordinary method.

(4) purifying of the fermentation of Yeast engineering bacterium strain and reorganization HD5 mutant polypeptide

Get-70 ℃ of frozen yeast engineering bacterial classifications that are integrated with pPIC9K-HD5-E21/R and be inoculated in the dull and stereotyped 30 ℃ of incubated overnight activation of YPD, the single colony inoculation of selecting good appearance is in the BMGY substratum, 30 ℃ of constant temperature shaking table 220rpm shaking culture 16～18h to OD ₆₀₀≈ 3～5, changeed to plant being cultured to OD 1 time then by 1: 10 ₆₀₀Being about 4, is seed liquor with this bacterium liquid.Then the seed liquor culture transferring in being housed, basic salt culture medium (is added trace salt PTM ₁) 15L fermentor tank (B.Braun company, Germany) carry out high-density culture fermentation.Leavening temperature is controlled at 30 ℃ (being controlled to be 28 ℃ behind the beginning methanol induction), and dissolved oxygen is controlled between 30%～40%, and the pH value is controlled at 5.0 (being controlled to be 3.5 behind the beginning methanol induction), and cell cultures is to OD ₆₀₀Begin stream during ≈ 150 and add methanol induction, methyl alcohol stream rate of acceleration is controlled at about 1%.Stop fermentation behind the abduction delivering 60h, low-temperature centrifugation is got supernatant, carries out purifying subsequently.At first supernatant is carried out the ultrafiltration chromatography,, and, use 20mmol/L phosphate buffered saline buffer (pH value 7.2) exchange buffering system simultaneously concentrated about 100 times of desired polypeptides with removal partial pigment and the high-intensity salt ionic concentration of reduction.Follow SP Sepharose Fast Flow post and carried out cation-exchange chromatography; Remove most residual pigments and foreign protein; Collection contains the component of desired polypeptides; Go up FINELINE post (Source RPC 15 fillers, the high 5cm of post) again and carry out reversed phase chromatography, will collect separated portion frozen purified recombinant HD5 mutant polypeptide that promptly obtains of substep in-80 ℃ of Freeze Drying Equipments.

The CTA of embodiment 3 HD5 mutant polypeptides

Inoculation vero cell (African green monkey kidney cell) is in 96 orifice plates; Inoculum density is 2000 cells/well, and the inoculation volume is 100 μ l, and the vero cell is cultivated 24h in the RPMI-1640 that contains 10% foetal calf serum adherent fully to cell; Abandon nutrient solution; Use the nutrient solution that contains different concns HD5 mutant polypeptide instead and continue to cultivate, each concentration all repeats 5 holes, and maternal HD5 polypeptide control group and blank group are set simultaneously.After cultivating 72h, every hole adds CCK-8 reaction solution 10 μ l, and 37 ℃ are continued to cultivate 4h, and inhale gently and abandon supernatant, the vibration mixing, ELIASA is measured 490nm wavelength absorbance (A value).Cell survival rate (%)=drug group A value/blank group A value * 100%.The result shows; 100 μ g/ml with interior HD5 mutant polypeptide can the remarkably influenced cell proliferation activity; And the cell survival rate between the HD5 mutant polypeptide treatment group of same concentrations and the maternal HD5 polypeptide treatment group do not have significant difference, shows that the cytotoxicity of HD5 two mutants does not increase.

The antiviral activity of embodiment 4 HD5 mutant polypeptides is measured

At first ordinary method prepares HSV-2 virus liquid, and measures the median infective dose (TCD of virus ₅₀).Inoculation vero cell is in 96 orifice plates, and inoculum density is 2000 cells/well, and the inoculation volume is 100 μ l, and the vero cell is cultivated 24h in containing the DMEM nutrient solution of 10% foetal calf serum adherent fully to cell, abandons nutrient solution, changes to contain 100 times of TCD simultaneously ₅₀The DMEM nutrient solution of virus liquid and 100 μ g/ml HD5 mutant polypeptides; Serum-concentration in the nutrient solution may be selected to be 0%, 2%, 10%, 20%; The normal cell control group is set simultaneously (neither adds viral liquid; Do not add the HD5 mutant polypeptide yet), virus control group (only add viral liquid, do not add the HD5 mutant polypeptide) and maternal HD5 polypeptide treatment group (add 100 times of TCD simultaneously ₅₀The maternal HD5 polypeptide of virus liquid and 100 μ g/ml).Behind 37 ℃ of cultivation 48h, every hole adds CCK-8 reaction solution 10 μ l, and 37 ℃ are continued to cultivate 4h, and inhale gently and abandon supernatant, the mixing that vibrates, ELIASA is measured 490nm wavelength absorbance (A value).Cytoprotective rate=(drug group A value-virus control group A value)/(normal cell control group A value-virus control group A value) * 100%.The result shows; Concentration is that the HD5 mutant polypeptide of 100 μ g/ml is no matter under the situation that has serum-free to exist; All can significantly protect the vero cell not infected by HSV-2, especially under there was situation in serum, its protection effect significantly was better than maternal HD5 polypeptide (seeing Fig. 1-4).

Conclusion: HD5 two mutants of the present invention not only has efficiently, the anti-HSV-2 virus capable of low toxicity; And the influence that not existed by serum of its antiviral activity, thereby make this mutant polypeptide not only can be used as externally applied agent but also can be used as the control that innerlich anwenden is applied to diseases of viral infection such as HSV, HPV and HIV.

Sequence table

< 110>Military Medical Univ No.3, P.L.A

< 120>people's α-alexin 5 antiviral active mutant polypeptides and preparation method and application

<130>

<160>3

<170>PatentIn version 3.3

<210>1

<211>32

<212>PRT

< 213>aminoacid sequence of people's α-alexin 5 antiviral active mutant polypeptides

<400>1

Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser

1 5 10 15

Leu Ser Gly Val Cys Arg Ile Ser Gly Arg Leu Tyr Arg Leu Cys

20 25 30

Cys Arg

<210>2

<211>96

<212>DNA

< 213>dna sequence dna of coding power people α-alexin 5 antiviral active mutant polypeptides

<400>2

gccacctgct attgccgaac cggccgttgt gctacccgtg agtccctctc cggggtgtgt 60

cgtatcagtg gccgcctcta cagactctgc tgtcgc 96

<210>3

<211>99

<212>DNA

< 213>dna sequence dna of the full-length gene of coding HD5 two mutants

<400>3

gccacctgct attgccgaac cggccgttgt gctacccgtg agtccctctc cggggtgtgt 60

cgtatcagtg gccgcctcta cagactctgc tgtcgctga 99

Claims

1. people's α-alexin 5 antiviral active mutant polypeptides, it is characterized in that: said mutant polypeptide hydrogen base acid sequence is following:

Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser

1 5 10 15

Leu Ser Gly Val Cys Arg Ile Ser Gly Arg Leu Tyr Arg Leu Cys

20 25 30

Cys Arg。

2. prepare the method for said people's α-alexin 5 antiviral active mutant polypeptides of claim 1, it is characterized in that: described preparation method is the solid state chemistry synthesis method.

3. the dna sequence dna of coding claim 1 described people's α-alexin 5 antiviral active mutant polypeptides, it has following sequence:

GCC ACC TGC TAT TGC CGA ACC GGC CGT TGT GCT ACC CGT GAG TCC CTC TCC GGGGTG TGT CGT ATC AGT GGC CGC CTC TAC AGA CTC TGC TGT CGC。

4. the method for preparing said people's α-alexin 5 antiviral active mutant polypeptides of claim 1; It is characterized in that: the dna sequence dna of described people's α-alexin 5 antiviral active mutant polypeptides of claim 3 is cloned in the expression vector; Transfection host cell; Through abduction delivering, purifying, promptly get said HD5 mutant polypeptide.

5. preparation method according to claim 4 is characterized in that: described expression vector is a plasmid expression vector.

6. preparation method according to claim 4 is characterized in that: described host cell comprises prokaryotic cell prokaryocyte and eukaryotic cell.

7. preparation method according to claim 6 is characterized in that: said prokaryotic cell prokaryocyte is a Bacillus coli cells.

8. preparation method according to claim 6 is characterized in that: said eukaryotic cell is a yeast cell.

9. said people's α-alexin 5 antiviral active mutant polypeptides of claim 1 are preparing the purposes of preventing and treating in hsv (HSV) infection or human papilloma shape virus (HPV) infection or human immunodeficiency virus (HIV) infection medicine.